Current microfluidic devices (“chips”) and systems allow for semi-automated and automated manipulation of small volumes, generally a few nanoliters. The miniaturization of sample processing and assaying provides for both increased speed and sensitivity, as well as an economy of scale—less reagent, sample and space is required to run the assay and obtain the desired information, and the decreased handling of individual samples and reagents reduces opportunity for error. Microfluidic devices and systems have been adapted, or proposed for use with a variety of chemical and biochemical analyses including protein crystallography, cell free protein synthesis, gas chromatography, cell separation, electrophoresis, polymerase chain reaction (PCR) and the like.
The advent of PCR and other nucleic acid-based methodologies, and completion of the sequencing of the human genome has led to various nucleic acid based diagnostics ant tests which continue to demand more precise and sensitive analytical tools. Knowledge of gene expression, polymorphisms, mutations (heritable or otherwise) and the like have been translated into improvements in health care—this in turn has demanded more precise and sensitive analytical tools. Earlier detection and diagnosis of genetic disease, cancer and infection provides immediate beneficial impact—early stages of disease may be treated more efficiently, and often with a greater degree of success, thus greatly improving subject outcome and quality of life.
Quantitative real-time PCR (RT-PCR) is a current ‘gold standard’ for detection of relatively rare polymorphisms, however it requires a difference of ˜20% or greater to be present in the sample before the difference is reliably detected. As PCR is an exponential technique, it is inherently very sensitive and in principal allows for the detection of single molecules. However, any non-specific amplification or contamination may lead to false positives, thereby making it very difficult to reliably detect rare sequences. This is particularly true when target sequences are very similar to other species that may be present at higher levels, and thus may limit the sensitivity of an assay in detecting rare molecular species—the presence of a single nucleotide polymorphism in rare population of cancer cells may be invisible due to high background of normal molecules with nearly identical sequence. Additionally, the real-time monitoring of an exponential reaction has high dynamic range but limited discriminatory power—differences below approximately 20% in the relative abundance of two sequences may be difficult to detect. In many applications a far greater sensitivity may be required. As an example, the detection of fetal aneuploidy from circulating blood would require accurate detection of allelic differences of approximately 1-6% Lo et al., 1998. Am J Human Genetics).
Digital PCR was initially described by Vogelstein and Kinzler in 1999 (Proc. Natl Acad. Sci USA 96:9236-9241). Digital PCR techniques provide for amplification of single molecules, however macroscopic implementations in microliter volume reactors using conventional 96 and 384 well plates may have to overcome non-specific amplification, contamination, high reagent costs, and modest numbers of reactions. Performing quantitative analysis by digital PCR therefore requires the reliable amplification of single molecules with low false positive rates—something that typically requires careful optimization in microliter volume reactors. In addition, the precision of analysis by digital PCR is dependant on the number of reactions. Reliable detection of a 1% difference would require hundreds of thousands to millions of reactions. This is not practical using existing methods.
Emerging microfluidic technologies provide increased sensitivity through small volume compartmentalization, high scalability, and economy of scale, thereby allowing for the full power of digital PCR to be realized.
Scaling down the size of an assay brings more than just an economy of scale—smaller samples may be used and more tests run on one sample is the most obvious benefit, however biological assays that were impractical, or not possible using ‘conventional’ volumes and samples sizes may also be enabled. In some cases, the conventional assays may simply be scaled down—for example use of 1 ul volumes instead of 100 ul; while in other cases, significant modification may be needed, in either the way the assay is set up, the data analysed or in other respects. Zhang et al., 2006 (Biotechnology Advances 24:243-284) reviews PCR microfluidic devices for DNA amplification and various methods, materials and techniques that may lend themselves to microfluidics applications.
The compartmentalization of solutions into a large number of small-volume reactions is useful in many fields. Microfluidic devices comprising valves and fluid channels allow for a planar (2-dimensional) emulsion to be formed with a regular spatial arrangement. This regular array has the advantage that it is possible to track or image each droplet over time. This is particularly important in assays that require time-monitoring of a readout (for example real time polymerase chain reaction). Furthermore, such methods allow for precisely defined arrays in which every drop has substantially the same volume.
Microfluidic digital PCR using compartmentalization by valves has been employed in multigene analysis of environmental bacteria (Warren et al., 2006. PNAS 103:17807-17812), and for transcription factor profiling of hematopoetic cells (Ottesen et al, 2006. Science 314:1464-1467). The microfluidic devices used in these experiments provide a compartmentalized array of approximately 9000 individual 10 nL reactions.
The achievable density and minimum volume of PCR reactions in current microfluidic devices may be subject to practical limitations. As the density of the array is increased, the volume of the individual reaction chambers is decreased, and valves may occupy too much space to be feasible. A maximum density at which microfluidic valves may be reliably fabricated (typically ˜2500 cm2, based on a 50×50 chamber array with a pitch of about 200 μm provides an upper limit to the number of individual valve-defined reaction chambers that may be fabricated (Thorsen et al., 2002. Science 298:580-584 and 2) the minimum volume of a PCR reaction that can be implemented in a gas permeable elastomer device without excessive reagent evaporation during thermocycling (˜1 nL). Density and scale are particularly important in digital PCR since accurate measurements require the compartmentalization of a single sample into thousands to millions of individual reactions, making it expensive in terms of both device area and reagent consumption. New methods for dramatically increasing assay density and reducing assay volume are therefore the central issue in realizing the full potential of this technique.
While a variety of materials are known and used in microfluidics applications, silicone rubber materials (e.g. polydimethylsiloxane, or PDMS) are preferred for the ease of handling and suitability to monolithic construction of microfluidic devices, PDMS exhibits a high gas permeability, making it possible to fill dead-end structures. In addition, PDMS has a high permeability to water vapour. Thus, some processes that are carried out in PDMS-constructed microfluidic devices—particularly where elevated temperatures are required, such as PCR may suffer from drying out of the small aqueous reaction volume due to rapid evaporation, and the reactions may fail.
Attempts to overcome this limitation have employed the use of external hydration methods. While this manages to reduce evaporation rates somewhat, the sample volume sizes that are able to be used successfully are still limited by evaporation even using this hydration. This limits the ability of such systems to incorporate very small volume sample sizes (e.g. on the order of picoliters or smaller) that would enable high density arrays—and thus large sample numbers to improve the utility of the devices.
U.S. Pat. No. 7,118,910 discloses a microfluidic device for performing PCR assays, and further discloses use of fluid-filled guard channels in a microfluidic device to reduce evaporation of fluid from the device.
U.S. Pat. No. 6,555,389 discloses a method for compensation for evaporation in a microfluidic device by replenishing fluid lost by evaporation from a reservoir of fluid, via capillary channels.